The goal of this proposal is to define biochemical and structural aspects of HIV-1 RT. This will involve identification of amino acid residues, and protein domains that are involved in reverse transcription. The recently solved 3D crystal structure of HIV-1 RT as an enzyme-DNA-dNTP ternary complex suggests a number of amino acids that may be involved in specific enzymatic steps. These residues will be modified by site-directed mutagenesis, and biochemical properties of the mutants will be characterized to determine their functional roles in catalysis. Mutagenesis will include amino acids along the DNA binding track to determine the influence of these residues on RNAse-H and strand transfer reactions. Deletion/insertion and point mutations in the p51 subunit will determine their roles in catalysis and in facilitating loading of p66 onto the template primer. The effect of these mutations on viral replication and infectivity will be determined. These studies will help to define the side chain orientation of amino acids in the catalytic domain. The assignment of functional significance to amino acids within the 3D structure of RT will promote better understanding of the mechanisms of RT catalyzed DNA synthesis, inhibitory action of anti-RT drugs, and structural basis for drug resistance. The specific aims are: 1). to determine the functional role of p51 in reverse transcription, 2). to determine the functional role of specific amino acids in RT catalysis, 3). to determine the in vivo effect of RT mutations on HIV-1 replication/infectivity, 3). to determine the correlation between mutational hotspots and the architecture of HIV-1 genomic RNA